Functional biomaterial research has been directed toward the development of improved scaffolds for wound healing and tissue engineering. A number of biodegradable polymers have been explored as scaffolds for wound healing and tissue engineering applications and include synthetic polymers like poly-caprolactone, poly(lactic-co-glycolic acid), poly(ethylene glycol), and natural polymers such as alginate, gelatin, collagen, starch, and chitosan. Among them, naturally derived polymers are of special interest due to, as natural components of living structures, their biological and chemical similarities to natural tissues. In this context, chitosan has been found as a fascinating candidate in a broad spectrum of applications along with unique biological properties including biocompatibility, biodegradability to harmless saccharide products, nontoxicity, physiological inertness, remarkable affinity to proteins, in addition to antibacterial, antifungal, and haemostatic properties.
The recorded use of chitosan dates back to the 19th century, when Rouget discussed the deacetylated form of chitosan in 1859. Chitin, the source material for chitosan, is one of the most abundant organic materials, being second only to cellulose in the amount produced annually by biosynthesis. It is an important constituent of the exoskeleton in animals, especially in crustaceans, molluscs and insects. It is also the principal fibrillar polymer in the cell wall of certain fungi, and can be produced by microalgae. Deacetylated chitin derivatives have been referred to as “chitosan”. When these two terms were first used in the 1800's, it was believed that chitin and chitosan always occurred in nature as distinct, well-defined, unique, and invariant chemical species, with chitin being fully acetylated and chitosan being fully deacetylated compositions. It was approximately a century later, however, before it was discovered that the terms “chitin” and “chitosan” are, in fact, ambiguous. Rather than referring to well-defined compounds, these terms actually refer to a family of compounds that exhibit widely differing physical and chemical properties. These differences are due to the products' varying molecular weights and varying degrees of acetylation.
Chitosan is a linear polysaccharide, composed of glucosamine and N-acetyl glucosamine units linked by β (1-4) glycosidic bonds—in essence, strings of sugar units. Depending on the source and preparation procedure, its molecular weight generally ranges from 10 kDa to over 1000 kDa. The molecular weight of the chitosan polymer is routinely determined by viscosity and is expressed in terms of Centipoise (CPS) or Millipascal (mPas) units, and can range from about 5 mPas to 3000 mPas. The content of glucosamine is termed as the degree of deacetylation (DD), and can range from 30% to 95%. In its crystalline form, chitosan is normally insoluble in aqueous solution above pH 7, however, in dilute acids (pH 6.0), the protonated free amino groups on glucosamine facilitate solubility of the molecule (Kim, Seo et al. 2008). Generally, chitosan has three types of reactive functional groups, an amino group as well as both primary and secondary hydroxyl groups at the C(2), C(3), and C(6) positions, respectively. These groups allow modification of chitosan for specific applications, which can produce various useful scaffolds for tissue engineering applications. The chemical nature of chitosan in turn provides many possibilities for covalent and ionic modifications which allow extensive adjustment of mechanical and biological properties.
Chitin Processing
As mentioned above, chitin is present within numerous taxonomic groups. However, commercial chitins are usually isolated from marine crustaceans, such as shrimp. Crustacean shells consist of 30-40% proteins, 30-50% calcium carbonate, and 20-30% chitin and also contain pigments of a lipidic nature such as carotenoids (astaxanthin, astathin, canthaxanthin, lutein and β-carotene). These proportions vary with species and with season.
When chitin is extracted by acid treatment to dissolve the calcium carbonate followed by alkaline extraction to denature and dissolve the proteins and by a depigmentation step, a colorless to off-white product is obtained mainly by removing the astaxanthin. The preparation method is a factor that affects the sample characteristics. Early studies have clearly demonstrated that specific characteristics of these products (Mw, DD) depend on the process conditions. Typically, however, commercial chitins are prepared by a first step of deproteinisation followed by a second step of demineralization. In these conditions a “collapsed chitin”, in which the native structure of the chitin is lost, is extracted. On the other hand, “compacted chitin”, in which the native chain and fibrous structures are intact and stabilized, is extracted when demineralization occurred in the first step. Chitosan prepared by either method of chitosan extraction apply to the present invention. Furthermore, the present invention does not restrict the source of chitosan from natural, semi-synthetic, or synthetic sources.
Chitin Deacetylation to Chitosan
Chitosan is prepared by hydrolysis of the acetamide groups of chitin. This is normally conducted by harsh alkaline hydrolysis treatment due to the resistance of such groups imposed by the trans arrangement of the C2-C3 substituents in the sugar ring (Horton and Lineback 1965).Thermal treatments of chitin under strong aqueous alkali are usually needed to give partially deacetylated chitin (DD higher than 70%), regarded as chitosan. Usually, sodium or potassium hydroxides are used at a concentration of 30-50% w/v at high temperature (100° C.). This harsh hydroxide/heat method has the coincident effect of reducing or removing potential bacterial endotoxins, which is beneficial for biomedical applications of the resulting chitosan materials.
Chitosan DD can range from 56%-99% depending on chitin source and methods of chitosan preparation (Abou-Shoer 2010). Factors that affect the extent of deacetylation include concentration of the alkali, previous treatment, particle size and density of chitin. In practice, the maximal DD that can be achieved in a single alkaline treatment is about 75-85% (Roberts 1998). In general, during deacetylation, conditions must be the proper ones to deacetylate, in a reasonable time, the chitin to yield a chitosan that is (subsequently) soluble in diluted acetic acid. It has become evident that the overriding factor regarding the fine structure of chitosan is the chemical polydispersion of the DD value (Roberts 1998). During chitosan deacetylation, the degradation of the polymeric chain takes place. Chitosan scaffolds with low DD (75-85%) displayed a more regular structure and the pores were fairly uniform and parallel with a polygonal cross section (Tigli and Gumusderelioglu 2008). The lateral pore connectivity was much lower than for scaffolds with high deacetylation degrees (>85%). Swelling studies were also performed but no relationship was found between DD and swelling ratio. Mechanical testing of chitosan scaffolds showed that mechanical strength was higher with higher DD. Biodegradability of the scaffolds also depends on the DD.
Chitosan Depolymerization
The main limitations in the use of chitosan in certain applications are its high viscosity and low solubility at neutral pH. Low Mw chitosans and oligomers can be prepared by hydrolysis of the polymer chains. For some specific applications, these smaller molecules have been found to be much more useful. Chitosan depolymerization can be carried out chemically, enzymatically, or physically. Chemical depolymerization is mainly carried out by acid hydrolysis using HCl or by oxidative reaction using HNO2 and H2O2. It has been found to be specific in the sense that HNO2 attacks the amino group of deacetylated glucosamine units, with subsequent cleavage of the adjacent glycosidic linkage (Prashanth and Tharanathan 2007). In the case of enzymatic depolymerization, low molecular weight chitosan with high water solubility were produced by several enzymes such as chitinase, chitosanase, gluconase and some proteases. Non-specific enzymes including lysozyme, cellulase, lipase, amylase and pectinase that are capable of depolymerizing chitosan are known. In this way, regioselective depolymerization under mild conditions is allowed (Aranaz, Mengibar et al. 2009).
There is no correlation between the Mw of chitosan and its swelling behavior (Roldo, Hornof et al. 2004; El-Kamel, Ashri et al. 2007). The tensile strength (TS), the percentage elongation at break (% EB) and the elastic modulus (EM) are important parameters to indicate the strength and elasticity of a film. ASTM International standard test methods have been established for the evaluation of the physical parameters for thin films or membranes (ASTM 2002; ASTM 2006). Medium Mw chitosan films have the highest values for TS and EM, followed by high Mw and low Mw chitosan films (El-Kamel, Ashri et al. 2007). On the other hand, the highest % EB is obtained for low Mw chitosan films, followed by high and medium Mw chitosan films.
Effect of Pore Variations
The mechanical properties of chitosan-based scaffolds are dependent on the pore sizes and pore orientations. Chitosan can be formed as interconnected-porous structures by freezing and lyophilizing a chitosan solution or by processes such as an “internal bubbling process (IBP)” where CaCO3 is added to chitosan solutions to generate chitosan—CaCO3 gels in specific shapes by using suitable molds (Chow and Khor 2000). Tensile testing of hydrated samples shows that porous chitosan membranes have greatly reduced elastic moduli (0.1-0.5 MPa, wherein a megapascal unit=N/mm2) compared to non-porous chitosan membranes (5-7 MPa). The extensibility (maximum strain) of porous membranes varied from values similar to nonporous chitosan (approximately 30%) to greater than 100% as a function of both pore size and orientation. Porous membranes exhibited a stress-strain curve typical of composite materials with two distinct regions: a low-modulus region at low strains and a transition to a 2-3 fold higher modulus at high strains. The tensile strengths of these porous structures are reportedly in the range of 30-60 kPa (Madihally and Matthew 1999).
Chen and Hwa reported effects of the molecular weight of used chitosans and their crystallinity on the mechanical property of chitosan membrane (Chen and Hwa 1996). That is, the lower molecular weight of chitosan used, the lower the tensile strength of the chitosan membrane prepared due to the chance of entanglement differences. In other words, the use of lower molecular weight chitosan produces less entanglement. Crystallinity difference of chitosan may be attributed to another factor. The lower the molecular weight of chitosan used, the lower the enthalpy of the resulting membrane. These implied that the lower tensile strength of the membrane was a result of less crystallinity in the chitosan membrane prepared from low molecular weight of chitosan.
Biodegradability
Chitosan is absent from mammals but can be degraded in vivo by several enzymes, most notably lysozyme, chitinase, and NAGase (Dalian, da Luz Moreira et al. 2007; Kim, Seo et al. 2008) (Aranaz, Mengibar et al. 2009) (Niekraszewicz 2005). Biodegradation leads to the release of non-toxic oligosaccharides of variable length which can be subsequently incorporated into glycosaminoglycans and glycoproteins, to metabolic pathways, or be excreted. Lysozyme, a non-specific glycoside hydrolase present in mammalian tissues and implicated in innate immunity, seems to play a significant degradation role on chitin and chitosan. The degradation kinetics seem to be inversely related to the degree of crystallinity, which is controlled mainly by the DD. Moreover, the distribution of acetyl groups also affects biodegradability since the absence of acetyl groups or their homogeneous distribution (random rather than block) results in very tow rates of enzymatic degradation.
Finally, several studies reported that the length of the chains (Mw) also affects the degradation rate. The understanding and control of the degradation rate of chitosan-based materials and medical devices is of great interest since degradation is essential in many small and large molecule release applications and in functional tissue regeneration applications. In certain uses, the rate of scaffold degradation should mirror the rate of new tissue formation or be adequate for the controlled release of bioactive molecules (e.g., natural compounds, pharmaceuticals, biologics, nucleic acids, vaccines, and immune effectors). Thus, it is important to understand and control both the mechanism and the rate by which each material is degraded.
The degradation rate also affects the biocompatibility since very fast rates of degradation liberate (and potentially accumulate) the amino sugars that can produce a mild inflammatory response. Chitosan samples with tow DD induce a more acute inflammatory response while chitosan samples with high DD induce a minimal response due to the low degradation rate. Degradation has been shown to increase as DD decreases. In other words, in general, degradation is enhanced by increased acetylation (Lim, Song et al. 2008). Kofuji et al. investigated the enzymatic behaviors of various chitosans by observing changes in the viscosity of chitosan solution in the presence of lysozyme (Kofuji, Qian et al. 2005). They found that chitosan with a low DD tended to be degraded more rapidly. However, other authors reported that differences in degradation are due to variations in the distribution of acetamide groups in the chitosan molecule. This occurs due to differences in deacetylation conditions which influences viscosity of the chitosan solution by changing the inter- or intra-molecular repulsion forces. Therefore, it can be concluded that it is impossible to estimate biodegradation rate from the DD alone.
Biocompatibility
Chitosan shows very good biocompatibility, but this property depends on the characteristics of the sample (e.g., natural source, method of preparation, Mw and DD). Although the digestive (oral/gastrointestinal) enzymes can partially degrade chitosan, when orally administered it is not absorbed. For this reason, chitosan is considered as not bioavailable by the oral route. Chitosan has a LD50 in mice of around 16 g/kg, a very high dose and consistent with negligible acute toxicity. Toxicity of chitosan is reported to depend on DD. Schipper et al. reported that chitosans with DD higher than 35% showed low toxicity, while a DD under 35% chitin) caused dose-dependant toxicity (Schipper, Varum et al. 1996). On the other hand, Mw of chitosan did not influence toxicity (Schipper, Varum et al. 1996).
The cytocompatibility of chitosan has been proven in vitro with myocardial, endothelial and epithelial cells, fibroblasts, hepatocytes, chondrocytes, and keratinocytes (Aranaz, Mengibar et al. 2009). This property seems to be related to the DD of the samples. When the positive charge of the polymer increases, the interactions between chitosan and the cells increase too, due to the presence of free amino groups. The adhesion and proliferation of keratinocytes and fibroblasts on several chitosan films with different DDs depend on both, DD and cell type. In both cells, the percentage of cell adhesion was strongly dependent on the DD, increasing with this parameter. The type of cell was a factor that also affected the adhesion, being more favorable for fibroblasts, which exhibit a more negative charge surface than for keratinocytes. On the other hand, the proliferation decreased considerably by increasing the DD. Therefore, a balance of cell adhesion and cell proliferation in wound healing and biological application requires an appropriate DD.
Chitosan films containing different Mw chitosans had different forces of adhesion but statistical analysis revealed that there was no significant difference in bioadhesion force between the films. On the contrary, Roldo et al. showed that the maximal detachment force of medium Mw chitosan was higher than that of both tow and high Mw chitosans (Roldo, Hornof et al. 2004).
Impure chitin and chitosan with residual proteins could cause allergic reactions such as hypersensitivity within some individuals. The protein content in a sample depends on the source of the sample and, especially, on the method of preparation. When prepared as described above (e.g., acid followed by strong base plus heat), purified chitosan is non-allergenic. While 0.2-0.3 percent of the human population exhibits allergies to marine crustaceans (Osterballe, Hansen et al. 2005; Osterballe, Mortz et al. 2009), the following conclusions were drawn from a respected authority on chitosan, Dr. Riccardo Muzzarelli:                It is presently unwise to interpret chitin as an allergenic substance, more clinical and genetic research being needed. Crab, shrimp, prawn and lobster chitins, as well as chitosans of all grades, once purified, should not be considered as “crustacean derivatives” because the isolation procedures have removed proteins, fats and other contaminants to such an extent as to permit to classify them as chemicals regardless of their origin. [(Muzzarelli 2010) p. 305]        The major shrimp allergen has been identified as the muscle protein tropomyosin . . . . Shrimp-derived glucosamine is safe even for individuals hypersensitive to tropomyosin. Villacis et al. state that glucosamine supplements from various manufacturers to not contain clinically relevant levels of allergens [76]. Gray et al. clearly state that “shellfish allergy is caused by IgE antibodies to antigens in the flesh of the shellfish and not the shell; therefore it should be safe for patients with shellfish allergy to take glucosamine supplement” [77]. [(Muzzarelli 2010) p. 300]        
Furthermore, with regard to purified chitosan as a material within “wound dressing” products, Dr. Muzzarelli states:                “In experimental and pre-clinical surgical trials, the use of chitin/chitosan and their derivatives has never led to allergies or other diseases.” [(Muzzarelli 2010) p. 304]Haemostatic Considerations        
Chitosan Mw also affects the binding or agglutination of red blood cells Shyu et al. 2001; Ishihara, Obara et al. 2006; Pang, Chen et al. 2007; Aranaz, Mengibar et al. 2009; Zhang, Xia et al. 2010). In a recent paper, a comparative study has been carried out among solid-state chitosan and chitosan acetic acid physiological saline solution (Jian, Feng et al. 2008). Several chitosan samples with Mw from 2000 to 400 kDa and DD from 90 to 70% were tested. It was found that solid-state chitosan and “chitosan acetic acid physiological saline solution” followed different haemostatic mechanisms. When blood was mixed with chitosan acetic acid physiological saline solution, the erythrocytes aggregated and they were deformed. The DD, especially a high DD, in the chitosan acetic acid physiological saline solution, had a significant effect on the unusual aggregation and deformation of erythrocytes, compared with the effect of Mw within a range between 100-1,000 kDa. However, this phenomenon could not be observed in solid-state chitosan. Solid-state chitosan with a high DD bound more platelets and was more haemostatic.
Numerous commercial medical device products containing chitosan and its salt forms are available for use in controlling hemorrhage (e.g., acidic lyophilized chitosan sponges). These devices are typically applied to the exterior surfaces of wounds as wound dressings or “bandages” (see below re: FDA Approved Devices)
Mucoadhesion
Several factors affect chitosan mucoadhesion, such as physiological variables and the physicochemical properties of chitosan. The mucus is composed of a glycoprotein called mucin, which is rich in negative charges since it has sialic acid residues. In the stomach, chitosan is positively charged due to the acidic environment and, therefore, it can interact with mucin by electrostatic forces. The extent of this union depends on the amount of sialic acid present in the mucin and on the Mw and DD of chitosan. It has been found that when the Mw of chitosan increases, the penetration in the mucin layer also increases and hence the mucoadhesion is stronger (Lehr, Bouwstra et al. 1992). On the other hand, a higher DD leads to an increase in charge density of the molecule and the adhesive properties become more relevant (He, Davis et al. 1998).
Antimicrobial Activity
One of the inherent properties of chitosan is that it confers considerable antibacterial activity against a broad spectrum of bacteria (No, Park et al. 2002; Jou, Yuan et al. 2007). Aimin et al. (Aimin, Chunlin et al. 1999) has shown that chitosan can reduce the infection rate of experimentally induced osteomyelitis by Staphylococcus aureus in rabbits. This is related to the cationic nature of chitosan by amino groups and to anions on the bacterial cell wall. The interaction between positively charged chitosan and negatively charged microbial cell wall leads to the leakage of intracellular constituents. The binding of chitosan with DNA and inhibition of mRNA synthesis occurs via the penetration of chitosan into the cytosol of the microorganisms and interfering with the synthesis of mRNA and proteins (Liu, Guan et al. 2001).
Other mechanisms have also been proposed. Chitosan may inhibit microbial growth by acting as a chelating agent rendering metals, trace elements or essential nutrients unavailable for the organism to grow at the normal rate. Chitosan is also able to interact with flocculate proteins, but this action is highly pH-dependent.
In addition, chitosan has antifungal properties. Several authors have proposed that the antimicrobial action of chitosan against filamentous fungi could be explained by a more direct disturbance of membrane function. However, it is not clear whether the antimicrobial activity of chitosan is caused by growth inhibition (fungistatic) or cell death (fungicidal).
Antioxidant Activity
Chitosan has shown a significant scavenging capacity against different radical species, the results being comparable to those obtained with commercial antioxidants. Samples prepared from crab shell chitin with DD of 90, 75, and 50% where evaluated on the basis of their abilities to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, superoxide, and alkyl radicals. The results revealed that chitosan with higher DD exhibited the highest scavenging activity (Park, Je et al. 2004). On the other hand, chitosans of different size as well as their sulfate derivatives, were assayed against superoxide and hydroxyl radicals. A negative correlation was found between chitosan Mw and activity. The chitosan sulfated derivatives presented a stronger scavenging effect on peroxide radicals but the chitosan of lowest Mw showed more considerable ferrous ion-chelating potency than others. The chelation of metal ions is one of the reasons why chitosan may be considered as a potential natural antioxidant. Chitosans may retard lipid oxidation by chelating ferrous ions present in the system, thus eliminating their pro-oxidant activity or their conversion to ferric ion (Peng 1998).
Current Uses of Chitosan
Chitosan, a natural cationic polysaccharide and salt forms thereof (e.g., -acetate, -lactate, -chloride, -phosphate, etc.) have received considerable attentions as a nontoxic and biodegradable biopolymer for diverse applications, especially in foods, medical devices, cosmetics and hair care products, and pharmaceutics (Johnson and Nichols 2000).
With regard to foods, in recent years chitosan was made available over the counter as a dietary supplement or cholesterol-lowering agent in multiple nutritional supplement products due to its ability to bind fat. Chitosans have been identified as versatile biopolymers of natural origin for food preservation due to their antimicrobial action against food spoilage microorganisms and antioxidant properties. The pH-dependent solubility allows them to be formed into various shapes (e.g., beads, films and membranes) using aqueous processing. Beads and particles have been described for use in resins, fillers, absorbants, adsorbants, and insulation (Smith 1994) (Unger and Rohrbach 1996). The use of chitosan coating as a protective barrier to extend the storability of many fruits and vegetables has been widely documented.
Current Medical Uses of Chitosan Structures
Due to its biological properties, chitosan has been employed in research and/or commercial products in wound healing management (e.g., wound dressings and “bandages”), implantable device systems such as orthopedic and periodontal composites, scaffolds for tissue regeneration, and drug- and DNA-delivery systems.
Chitosan, as a biodegradable natural biopolymer, has served as a biocompatible wound dressing for many years. Chitosan-based materials are highly biocompatible without toxicity and with only an early, mild, macrophage-dominated inflammatory response. In general, the unique chemical and biological properties, biodegradation characteristics, and biocompatibility of chitosan make it attractive in biomedical applications. Chitosan-containing products are currently available on the medical market, typically as US FDA Class I medical device wound dressings or “bandages” to promote wound healing. Chitosan-based products have been used perhaps even more extensively internationally than in the United States.
Purified Chitosan Safety in Humans
The safety of purified chitosan in humans has been widely reported (Illum 1998; Baldrick 2010). Safety in humans has been demonstrated in various contexts:
1. FDA Approved Devices:
Purified chitosan is a component in multiple US FDA-approved Class I medical and dental devices, and in most cases as the principal component. It has been used in various finished product forms, such as granules, a film component of bandages and gauze, and a lyophilized “sponge”. Examples of FDA 510(k) Premarket Notification cleared Class I products include HemCon Bandage, HemCon Dental Dressing, HemoHalt Hemostasis Pad Wound Dressing, Aquanova Super-Absorbent Dressing, CELOX Topical Hemostatic Granules in Soluble Bag, and ChitoGauze.
2. GRAS Food Additive:
Chitosan is considered as Generally Accepted as Safe (GRAS) as a food additive at the level of “self-affirmed” by various manufacturers of chitosan (e.g., Primex). To the best of our knowledge, a GRAS designation at the higher level of “no comment” following a full FDA review has not yet occurred. Chitosan is considered by the scientific community to be safe for use in foods, albeit with one caveat—ingested chitosan has affinity for dietary lipids and can reduce lipid uptake from the gastrointestinal tract.
3. Cosmetic & Consumer Skincare Products:
Chitosan is listed among the International Nomenclature of Cosmetic Ingredients (INCI). Chitosan and its various salt forms (e.g., lactate, glycolate, ascorbate, formate, & salicylate) and other organic derivatives are listed as ingredients for use in cosmetics and consumer skincare products, and via multiple vendors. However, chitosan has not yet undergone an evaluation by the Cosmetics Ingredients Review (CIR). This panel of industry experts evaluates a very limited number of cosmetic ingredients for safety. To the best of our knowledge, chitosan has not warranted consideration by the expert panel, and is considered by the scientific community as safe for use in consumer skincare and cosmetic products.
Tissue Engineering
Tissue engineering is a multidisciplinary science, including fundamental principles from materials engineering and molecular/cellular biology in efforts to develop biological substitutes for failing tissues and organs. In the most general sense, tissue engineering seeks to fabricate living replacement parts for the body. Langer and Vacanti (Langer and Vacanti 1993) reported that the most common approach for engineering biological substitutes is based on living signal molecules, and polymer scaffolds. The cells synthesize matrices of new tissue as well as function on behalf of the diseased or damaged tissues, while the scaffold provides the suitable environment for the cells to be able to effectively accomplish their missions such as adherence, proliferation, and differentiation. The function of the signal molecules is to facilitate and promote the cells to regenerate new tissue. The scaffolds provide not only temporary three-dimensional frameworks to form the designed tissues, but also space filling and controlled release of bioactive signal molecules. To perform these varied functions in tissue engineering, scaffold should meet the following requirements: (1) biocompatibility with the tissues, and an environment that promotes cellular adhesion, (2) biodegradability at the optimal rate corresponding to the rate of new tissue formation, (3) nontoxicity and non-immunogenicity, (4) optimal mechanical properties, and (5) adequate porosity and morphology for transporting of gases, metabolites, nutrients and signal molecules both within the scaffold and between the scaffold and the local environment.
Chitosan is one of the most promising biomaterials in tissue engineering because it offers a distinct set of advantageous physico-chemical and biological properties that qualify them for tissue regeneration in various kinds of organs such as skin, bone, cartilage, liver, nerve and blood vessel. Recent studies in regenerative tissue engineering suggest the use of scaffolds to support and organize damaged tissue because three-dimensional matrices provide a more favorable ambient for cellular behavior. Due to their low immunogenic activity, controlled biodegradability and porous structure, chitosan scaffolds are promising materials for the design of tissue engineered systems.
It is known that the microstructure such as pore size, shape and distribution, has prominent influence on cell intrusion, proliferation and function in tissue engineering. Cell attachment studies on the scaffolds showed that higher DD favored cell adhesion (Seda Tigli Karakecili et al. 2007). The present disclosure, however, contemplates chitosan DD from 56% to 99%.
The degradability of a scaffold plays a crucial role on the long-term performance of tissue-engineered cell/material constructs because it affects many cellular processes, including cell growth, tissue regeneration, and host response. If a scaffold is used for tissue engineering of the skeletal system, degradation of the scaffold biomaterial should be relatively slow, as it has to maintain the mechanical strength until tissue regeneration is completed or nearly so. The degradation rate also inherently affects both the mechanical and solubility properties over time.
Recently, attention has been focused on making polymeric nanofibers by electrospinning process as a unique technique because it can produce chitosan nanofibers with diameter in the range from several micrometers down to tens of nanometers, depending on polymer and processing conditions. Electrospinning applies high voltages to a capillary droplet of polymer solution or a melt to overcome liquid surface tension and thus enables the formation of much finer fibers than conventional fiber spinning methods. These nanofibers mimic the structure and function of the natural extracellular matrix (ECM) and are of great interest in tissue engineering as scaffolding materials to restore, maintain or improve the function of human tissue, because they have several useful properties such as high specific surface area and high porosity. The recent attempts have been made to prepare chitosan-based nanofibrous structures by electrospinning, with varying degrees of success. Min et al. (Min, Lee et at. 2004) produced chitin and chitosan nanofibers with an average diameter of 110 nm and their diameters ranged from 40 to 640 nm by the SEM image analysis. Bhattarai et al. (Bhattarai, Edmondson et al. 2005) further concluded that these chitosan-based nanofibers promoted the adhesion of chondrocyte and osteoblast cells and maintained characteristic cell morphology.
Wound Healing
Chitin and chitosan activate immunocytes and inflammatory cells (e.g., PMNs and macrophages), fibroblasts and angio-endothelial cells. These effects are related to the DD of the samples, chitin presenting a weaker effect than chitosan. Okamoto and coworkers reported that chitosan influenced all stages of wound repair in experimental animal models (Okamoto, Shibazaki et at. 1995). In the inflammatory phase, chitosan has unique hemostatic properties that are independent of the normal clotting cascades. In vivo these polymers can also stimulate the proliferation of fibroblasts and modulate the migration behavior of neutrophils and macrophages modifying subsequent repair processes such as fibroplasias and re-epithelialization (Okamoto, Shibazaki et al. 1995; Kosaka, Kaneko et al. 1996). Kosaka et al. reported that the cell binding and cell-activating properties of chitosan play a crucial role in its potential actions. These studies have added further to the body of evidence that chitosan is suitable as a wound healing material where cell-seeding onto chitosan-based scaffolds would provide tissue engineered implant being biocompatible and viable.
Chitosan oligomers have also exhibited wound-healing properties (Minagawa, Okamura et al. 2007). It is suggested that their wound-healing properties are due to their ability to stimulate fibroblast production by affecting the fibroblast growth factor. Subsequent collagen production further facilitates the formation of connective tissue (Howling, Dettmar et al. 2001).
The potential use of chitin oligosaccharides in wound healing as well as their capacity against chronic bowel disease has been studied (Deters, Petereit et al. 2008). The wound healing effect of chitosan oligomers and monomers is of great interest because in vivo lysozyme degrades chitosan polymer to these smaller molecules.
Chitosan-based implants have been found to evoke a minimal foreign body reaction, with le or no fibrous encapsulation. The typical course of healing is with formation of normal granulation tissue, often with accelerated angiogenesis. Chitosan possesses the properties favorable for promoting rapid dermal regeneration and accelerating wound healing suitable for applications extending from simple wound dressings to sophisticated artificial skin matrices. During the course of chitosan implant degradation by macrophage-like cells, the chitosan has been reported to stimulate an anti-inflammatory cytokine cascade (Chellat, Grandjean-Laquerriere et al. 2005).
An ideal cutaneous dressing would control the evaporative water loss from a wound at an optimal rate. The transepidermal water loss (TEWL) rate for normal skin is 204 g/m2 per day, while that for injured skin with a compromised stratum corneum and epidermis can range from 279 g/m2 per day for a “first-degree” burn to 5138 g/m2 per day for a granulating wound lacking the epidermis. The water vapor permeability of a wound dressing should prevent both excessive dehydration as well as buildup of exudate. It was recommended that a rate of 2500 g/m2 per day, which being in the mid-range of loss rates from injured skin, would provide an adequate level of moisture without risking wound dehydration. The water loss data for fabricated asymmetric chitosan membranes ranged from 2109 to 2792 g/m2 per day depending on the per-evaporation time before membrane casting (Mi, Shyu et al. 2001). The high porosity of the sponge-like sublayer increases the adsorption of water vapor and the decreased thickness of dense skin layer increases the diffusion of water molecule, thus resulting in the increased water vapor transmission rate.
Drug Delivery Systems
An important application of chitosans in industry is the development of drug delivery systems such as nanoparticles, hydrogels, microspheres, films and tablets. As a result of its cationic character, chitosan is able to react with polyanions giving rise to polyelectrolyte complexes. Pharmaceutical applications include nasal, ocular, oral, vaginal, parenteral, and transdermal drug delivery. Three main characteristics of chitosan to be considered are: Mw, DD, and purity. When chitosan chains become shorter (low Mw chitosan), they can be dissolved directly in water, which is particularly useful for specific biomedical applications, when pH should stay at around 7.0, or slightly lower (ca. 5.5-6.5) for dermatologic or consumer skincare applications.
In drug delivery, the selection of an ideal type of chitosan with certain characteristics is useful for developing sustained drug delivery systems, prolonging the duration of drug activity, improving therapeutic efficiency and reducing side effects. The physicochemical characteristics of chitosan are important for the selection of the appropriate chitosan as a material for drug delivery vehicles.
The DD controls the degree of crystallinity and hydrophobicity in chitosan due to variations in hydrophobic interactions which control the loading and release characteristics of chitosan matrices (Draget 1996). Zhang et al. also reported that a high chitosan DD and narrow polymer Mw distribution were shown to be critical for the control of particle size distribution (Zhang, Oh et al. 2004).
Desai and Park observed that the release rate of vitamin C was much lower as the Mw of chitosan used for preparing microspheres increased (Desai and Park 2006). They studied the release kinetics and found that it followed Fick's law of diffusion.
With regard to in vitro release studies, the amount of drug released is similar for films that contained low and medium Mw chitosan, but lower for the ones prepared with high Mw chitosan. This behavior is predictable, taking into account the direct relationship between the molar mass of chitosan and the viscosity of its solution. By increasing the viscosity of the polymer, the diffusion of the drug through the formed gel layer into the release medium was retarded (El-Kamel, Ashri et at. 2007).
Gene Delivery
Due to its positive charge, chitosan has the ability to interact with negatively charged molecules such as DNA. This property was used for the first time in 1995 to prepare a non-viral vector for a gene delivery system (MacLaughlin, Mumper et al. 1998). The use of chitosan as non-viral vector for gene delivery offers several advantages compared to viral vectors. Mainly, chitosan does not produce endogenous recombination, oncogenic effects and only mild immunological reactions. Moreover, chitosan/plasmid DNA complexes can be easily prepared at low cost.
The Mw of chitosan is a key parameter in the preparation of chitosan/DNA complexes since transfection efficiency correlates strongly with chitosan Mw. High molecular weight chitosan renders very stable complexes but the transfection efficiency is very low. To improve transfection efficiency, recent studies have examined the use of low Mw chitosans and oligomers in gene delivery vectors. It appears that a fine balance must be achieved between extracellular DNA protection (better with high Mw) versus efficient intracellular unpackaging (better with low Mw) in order to obtain high levels of transfection. Lavertu et al. studied several combinations of Mw and DD of chitosan finding two combinations of high transfection efficiency using a chitosan of 10 kDa and DD of 92 and 80%, respectively (Lavertu, Methot et al. 2006).
Kiang et al, studied the effect of the degree of chitosan deacetylation on the efficiency of gene transfection in chitosan-DNA nanoparticles (Kiang, Wen et al. 2004). Highly deacetylated chitosan (above 80%) releases DNA very slowly. They suggest that the use of chitosan with a DD below 80% may facilitate the release of DNA since it lowers the charge density, may increase steric hindrance in complexing with DNA, and is known to accelerate degradation rate. They reported an increase in luciferase reporter gene expression when the DD was decreased from 90% to 70%. Formulations with 62% and 70% deacetylation led to luciferase transgenic expression two orders of magnitude higher than chitosan with 90% deacetylation.
Chitosan Membranes
A potential and practical use for a chitosan membranes or films is as a barrier membrane to separate tissue layers during surgery. Three methods are typically used to produce membrane-like or film-like chitosan structures of low to high density. These preparation methods are solvent casting, phase separation, and immersion-precipitation phase inversion (Madihally and Matthew 1999; Hong, Wei et al. 2007). For all three methods, chitosan solutions of varying concentrations (e.g., 2-4% w/v) are prepared by dissolving the appropriate amount of chitosan powder (e.g., 75-90% DD/400-500 mPas) in a 1% (v/v) acetic acid solution. Next, the chitosan solution is cast into custom silicone mold cavities. At this point the three different methodologies, described below, diverge one from another.
In the phase separation method, the casted acidic chitosan solution is frozen at −20° C. overnight and then freeze dried at −40° C. at 10×10−3 mBar for 48 hours. The freeze-dried chitosan material is then de-molded and treated with 1N NaOH for 4 h to stabilize the chitosan polymer network, repeatedly washed with distilled water and then placed in a 50° C. oven for drying. The phase separation method results in a relatively low density porous “sponge” with a pore size that can be controlled (Mi, Shyer et al. 2001) (No et al. 2002).
Freezing of a chitosan solution produces two or more distinct phases—typically water freezing into ice with displacement of the chitosan biomaterial into a separate solid phase. Another step is required to remove the frozen solvent (typically ice), and hence produce the low-density porous sponge, which is a form commonly used in wound dressings. This is accomplished without disturbing the fibrous structure by a freeze-drying (i.e., lyophilization) and/or a freeze substitution step.
For the solvent cast method, the casted acidic chitosan solution is simply dried in an oven at 50° C. to remove the solvent, leaving a chitosan membrane. After drying, the chitosan membranes are treated with 1N NaOH for 4 h, repeatedly washed with distilled water to remove any traces of reacting agents and then placed in an oven at 50° C. for drying. As the solvent starts to vaporize after the solution is cast in this process, the solvent on the surface of the polymer solution vaporizes faster than that of the inside, so the concentration of polymer increases quickly to form the layer shaped by means of the colloid particle. After the forming of the surface layer, vaporizing of the solvent slows down. The chitosan solubility is not enough to maintain the system as a homogeneous solution and results in phase separation. Solvent separating from the homogeneous solution forms a polymer-poor phase surrounded by a polymer-rich phase. The exchange of acidic solvent with neutralizing base stabilizes the polymer network.
In a third approach, the immersion-precipitation phase inversion (IPPI) method, the casted acidic chitosan solution is (partially) dehydrated in an oven at 50° C. for 1 hour to form an asymmetric membrane, subsequently the chitosan polymer in the membrane is stabilized by immersion into a 0.2 M NaOH solution for 24 hours. The resulting membrane is then washed repeatedly with deionized water and then freeze-dried for 48 hours. The IPPI method results in an asymmetric porous membrane with three layers: a dense outer layer, a less dense middle transition layer and then a spongy porous layer, all of which can be controlled (Hong, Wei et al. 2007).
Reviews on chitosan and its uses have been published (Kato, Onishi et al. 2003; Niekraszewicz 2005; Boateng, Matthews et al. 2008; Aranaz, Mengibar et al. 2009; Zhang, Xia et al. 2010).
The making and use of chitosan sponges are described in the prior art:    (1) for uncompressed lyophilized neutralized sponge (Zhang, Cheng et al. 2006; Seda Tigli, Karakecili et al. 2007; Blan and Birla 2008); and    (2) for uncompressed lyophilized non-neutralized sponge (Tully-Dartez, Cardenas et al. 2010; McAdams, Block et al. 2011).
There are several other described methods to increase the density of chitosan materials including:    (1) Compression of a lyophilized acidic sponge to unspecified density (McCarthy, Gregory et al. 2008; Gregory and McCarthy 2009);    (2) Compression of a lyophilized acidic sponge to a specified density less than or equal to 0.8 g/cm3 (McCarthy, Gregory et al. 2008; Gregory and McCarthy 2010; McAdams, Block et al. 2011; McCarthy, Gregory et al. 2011);    (3) Asymmetric air drying (Ma, Wang et al. 2001; Thein-Han and Stevens 2004; Kuo 2005; Kuo, Chang et al. 2006; Dallan, da Luz Moreira et al. 2007; Duan, Park et al. 2007; Hong, Wei et al. 2007; Pang, Chen et al. 2007; Kuo 2008) (Ma et al. 2001) (Duan et al. 2007); and    (4) Electrospinning followed by rolling (Yeo, Jeon et al. 2005; Li and Hsieh 2006; Park, Kang et al. 2006). Electrospinning produces thin, neutralized chitosan fibers that can be blended together in a layered web product. Electrospinning technology does not apply to the present invention described herein.
Chitosan structures can be strengthened by cross-linking chemically with or without the requirement for light activation (Masuoka, Ishihara et al. 2005; Obara, Ishihara et al. 2005). However, none of these cross-linking methods can increase the density of chitosan to the high-density range of the present invention described herein.
Asymmetric air-drying increases the density of a chitosan solution by evaporation of acidic solvent from the exposed surface of the chitosan solution. As the solvent is removed, the density of the chitosan on the exposed surface increases. This method of increasing chitosan density can result in a dense, membrane-like chitosan device. A particular problem with this method is the uneven nature of surface evaporation of a solution within a mold, and the limited density that can be achieved without compression. An additional problem with manufacturing dense chitosan membrane structures by the use of air drying alone is that swelling of the dried membrane upon wetting is excessive and clinically problematic for materials intended as dense and thin barrier membranes. Therefore, unlike the prior art, the present invention describes a novel method of creating a high-density membrane-like chitosan material that circumvents current problems.